The Research Of Producing APP-BG Vaccine By Fermentation Technology

Actinobacillus pleuropneumoniae (APP) is a contagious disease, which can cause porcinecontagious pleuropneumonia. It is not only a infectious disease in world’s industrializedpig-breeding, but also a disease which could make a lot of loss in pig-breeding. With thereason of so many serotypes and weak multiple drug resistance, the traditional deactivationvaccine and weak poisonous vaccine have poor effect to porcine contagious pleuropneumonia.The BGV with high immunogenicity and adjuvant is a new prevention method to APP. Nowthe study of BG in China has limited in the laboratory. It has the basic characteristics of highaccuracy, low yield and high cost of production. The aim of this essay is to find a new methodto increase the production of BGV, lower the production cost and make fermentationindustrialized and large-scaled.In this experiment, we used pMC-WK APP I as a strain to explore fermentationtechnology to produce APP bacterial ghost vaccine. pMC-WK APPⅠis a recombinant strainwith host bacterium of APP Ⅰ, which contains temperature control genes of λ pL/pR-cI857,lysis-E and SNUC. Firstly, the experiment puts up the examination method of pMC-WK APPⅠand BG. It tests on pMC-WK APPⅠby gram stain, and review the PCR products ofpMC-WK APPⅠ plasmid amplified by specific primers of DLS. Though the stain and thepresence of a plasmid can be ascertained, PCR method can not test the BG of content afterlysis. So in order to test APP, the indirect ELISA is used to test outer membrane antigen. Themethod can test bacteria in OD600nm≥0.4, and test rate is more than84%. Before the studyof fermentation technology, it still need a further study in the lysis conditions of BG bypMC-WK APPⅠ. In laboratory, by the contrast experiment on the best temperature andopportunity, we found that the optimal ratio of lysis system, which firstly develop pMC-WKAPPⅠin28, and after OD600reached0.08~0.14, increase temperature to42℃for1.5h, andthen reincrease to47℃for2h. The key to lower the production costs is medium. So weoptimize the essential components and contents of high density producing culture in APP-BGvaccine by pMC-WK APPⅠ. By single factor test, we found that the content of yeast powder,soya peptone and glucose medium have a significant impact on BG-yield. We used responseSurface Methodology(RSM) to gain an interaction between the quadratic regression equationof an experimental yield on the three factors and BG-yield. The yield of APP-BG vaccinereaches the optimal value3.381and production cost is2.95RMB/g when yeast powder is10.86g/L, soya peptone is13.33g/L and glucose is2.58g/L. Trough the repeated experimentof inoculation quantity, stirring speed, dissolved oxygen and pH, the whole process offermentation comprises the following steps: seed culture, fermentation and BG harvest. In it, fermentation stage includes batch producing, induced lysis and completion in hightemperature. In batch producing stage, the inoculation quantity is controlled in2%,temperature is28℃、stirring speed is300rpm, dissolved oxygen concentration is20%and pHis7.2. After2h, when OD600reaches0.1~0.15, increase temperature to42℃for10min.Then there is no need to add anty substance to induce lysis for90min in42℃. At last increasetemperature to47℃for2h to complete high temperature stage. In SEM, the appearance of BGafter lysis is basically similar with the original one, but only outer membranes becomewrinkle. Though it was processed in47℃with shaking, the outer membrane protein amongBGV, APPⅠstain in high temperature and lysised in42℃are exactly same by SDS-PAGE,and their surface antigen are intact. At the same time, the protection rate of APP BGV couldbe achieved100%from APP in toxicity attack test.